Embryo and larval biology of the deep-sea octocoral Dentomuricea aff. meteor under different temperature regimes

Target species and colony collection

The Azores Archipelago, located above the Mid-Atlantic Ridge, is a biodiversity hotspot for deep-sea octocorals (Sampaio et al., 2019). Coral gardens (OSPAR, 2010) formed by deep-sea octocorals are among the most prominent deep-sea communities on regional seamounts and island slopes (Braga-Henriques et al., 2013). Dentomuricea aff. meteor is an octocoral species of the family Plexauridae, so far only recorded on the seamounts of the North Mid-Atlantic Ridge. It is common in regional seamounts between 200–600 m (Braga-Henriques et al., 2013), where it forms dense populations, often in combination with other octocoral species such as Viminella flagellum and Callogorgia verticillata (Tempera et al., 2012). The species is gonochoristic and presents gametes all year round, with seasonal peaks of gamete maturation and spawning usually occurring in autumn (M Rakka, 2020, unpublished data).

A total of 11 colonies of the species Dentomuricea aff. meteor were collected as by-catch from experimental long-line fisheries on board RV Archipelago (ARQDAÇO monitoring programme). Collection was performed at the summit of Condor Seamount, between 200–280 m, in September and October 2019. Colonies were divided in large fragments (20–30 cm height) and were kept at the DeepSeaLab aquaria facilities (Orejas et al., 2019), in six 33L aquaria positioned in a thermo-regulated room at 14 °C. Aquaria were supplied continuously with seawater (SW) pumped from 5m depth, previously treated with UV light (P10 UVsystem & Vecton 600 TMC™) and passed through 50 µm and 1 µm mesh filters. Circulation within the aquaria was maintained by pumps. Seawater temperature was kept between 13–14 °C with the aid of chillers and salinity was 35.8 ±0.1, similar to the natural conditions at the collection site (Santos et al., 2013). Colonies were fed twice per day with a mixture of frozen zooplankton and microplankton which was frequently enriched with live microalgae (Chaetoceros calcitrans and Nannochloropsis gaditana) and live rotifers.

Larval rearing

Larvae were obtained by maintaining reproductively active female and male colonies in the same aquaria to achieve natural spawning and fertilization. Coral fragments were allowed to acclimatize in the above aquaria conditions for approximately one month. Subsequently, colonies with mature gametes were identified by dissecting two branchlets (3–5 cm height) from each colony and observing their tissue under a dissecting microscope. Reproductively immature colonies and fragments in poor condition were excluded from further analysis. This procedure resulted in selection of six female and three male colonies. Coral fragments from the female colonies were distributed in two aquaria, referred to as spawning aquaria. Subsequently the fertile male colony with the higher number of available fragments was selected and four of its fragments were distributed in each of the two spawning aquaria. The remaining male colonies were not used to avoid polyspermy (Levitan, Terhorst & Fogarty, 2007).

To increase the potential of spawning, we enriched the aquaria water with free mature sperm, obtained from the selected male colony. This was achieved by dissecting mature spermatocysts from coral tissue, which were subsequently concentrated in 50 ml flasks with filtered (mesh size: 0.2 µm) SW, mixed by gently shaking and redistributed to the aquaria. Water inflow was paused and aquaria pumps were substituted with aeration to ensure water circulation without losing or harming potentially spawned gametes. Upon gamete release, which happened in batches separated by intervals of at least 2–3 h, gametes/fertilized eggs from each batch were collected from the water column to a 750 ml-culture flask (20–100 fertilized eggs per flask), filled with filtered SW from the aquaria facilities (mesh size: 0.2 µm). Whenever more than 100 gametes/embryos were released in one batch, these were equally distributed to two flasks to avoid maintaining larvae in high densities. During the first four days of the study we collected a total of 688 gametes which were distributed to 7 batches. Three of these batches were large enough to be split to two flasks (total n = 10 flasks).

Temperature experiments

In order to choose appropriate temperature regimes for larval rearing, we utilized temperature data collected during annual CTD surveys, under the framework of the projects CONDOR (EEA Grants PT-0040) and SMaRT (SRECC- Azores Regional Government M.2.1.2/029/2011). Data were collected between 2010 and 2012, above the coral garden where specimen collection took place. Subsequently, we utilized the minimum and maximum recorded values during the spawning season of the target species (October-November) to define the target rearing temperatures (13 ±0.5 °C and 15 ±0.5 ° C). Two water baths were set-up, each maintaining temperature within ±0.5 °C of the corresponding target temperature, with the aid of an aquaria chiller and a heater, respectively. Each day, the collected batches were divided between the two temperature treatments: immediately after collection of the released fertilized eggs/embryos, culture flasks were randomly assigned to one of the two water baths (n = 5 in each water bath). This corresponded to a total of 346 and 342 embryos reared at 13 °C and 15 °C respectively. Culture flasks were equipped with glass pipettes connected to an aquaria air pump, achieving continuous light circulation, while the full volume of water in the flasks was exchanged daily.

Embryonic and larval development

Embryos were monitored every 3–4 h during the first 48 h and subsequently once a day until reaching the planula stage, to study their early development. In every monitoring event, all embryos were counted to estimate survival. Additionally, 10–15 embryos were randomly removed from each flask and photographed, with a digital camera (DIGICAM 5MEG LCMOS MAC) attached to a microscope (10×), to record their developmental stage and size. Embryos were subsequently returned to the flasks. Due to the sometimes prolonged gamete release, gametes of the same batch were occasionally in slightly different developmental stages, therefore the timing of embryonic development is approximate. Moreover, since it was not possible to define the moment of fertilization, embryo development is presented in respect to the time of gamete release. To estimate size, we measured width and length (mm) of embryos and larvae (days 4 and 14) using the open software Fiji/Image J (Schindelin et al., 2012). The data were subsequently used to estimate volume (mm³) assuming larvae had the shape of a prolate spheroid (Larsson et al., 2014). The ratio of length to width (LW ratio) was used as a proxy of sphericity.

Embryo and larval survival

After reaching the planula stage, larvae were counted every 2–3 days. The last count corresponded to day 34, 36 or 39, depending on the batch. The obtained data were joined to the dataset collected during embryo development to estimate larval survival during the whole experimental period. Survival analysis was performed using the Kaplan–Meier method (Kaplan & Meier, 1958), following the rationale of Graham, Baird & Connolly (2008). Since monitoring was done in time intervals and the exact time of death for each larva was not known (interval-censored data), we assumed that time of death was the moment at which each larva was observed for the last time. The remaining larvae at the last monitoring event were considered alive (censored data). As the Kaplan–Meier method does not allow for incorporation of replicate information into the analysis, we performed the analysis by pooling data from all batches together, for each rearing temperature. Subsequently the analysis was repeated separately for each batch, to provide information about the variability among batches (Graham, Baird & Connolly, 2008). A log-rank test was performed to compare the survival curves between larvae reared under 13 °C and 15 °C. Survival analysis was performed by using the packages survival (Therneau & Grambsch, 2000) and survminer (Kassambara, Kosinski & Biecek, 2019) in R 3.5.0 (R Core Team, 2019).

Larval swimming behaviour

Data on swimming speed and behaviour were collected by video recording and analysis. Videos were recorded with a Canon EOS 600D digital camera, equipped with a regular 22–55 mm lens, on day 4 and day 15 after spawning, which corresponded to the first day larvae reached the mature planula stage and the second day larvae started settling, respectively. To minimize larval handling, swimming behaviour was recorded in the same culture flasks used for larval rearing. Videos were captured in the dark, using lateral led lights for illumination (Stromberg & Larsson, 2017). Flasks were positioned in front of a black slide with a calibrated grid that was used as background and a 2-minute waiting period was implemented to ensure no water movement was interfering with larval swimming. Subsequently, three videos (duration: 1 min) were recorded at three minute intervals.

Videos were converted to frames and were analyzed by an automatic particle tracking method, using the open software Fiji/Image J (Schindelin et al., 2012) and the plugin TrackMate (Tinevez et al., 2017) to record data on vertical swimming behaviour, namely swimming direction (up/down), displacement and swimming speed. Estimates of swimming speed only considered tracks with displacement higher than 2 mm, to exclude data from larvae that did not move or moved minimally.

Pelagic phase and larval settlement

During the counts performed for survival, each larva was assigned to one of four stages: planula, settled, pelagically metamorphosed and deformed. Because counts were made simultaneously for all flasks and each flask contained a batch of different age, e.g., some batches were released with 1–3 day difference, when average counts were estimated these were sometimes heavily influenced by the available count for that day. To be able to estimate robust mean counts for each monitoring day, missing counts were regenerated for each batch separately by using linear interpolation between existing data points (Dong & Peng, 2013), by using the R package VIM (Kowarik & Templ, 2016). Extrapolation was performed only until the last datapoint that was available for each batch, i.e., there was no attempt to predict the trend past the last available count. Subsequently, counts of each stage were divided by the total number of living larvae in each batch. This resulted in estimates of the proportion of the surviving larvae in each stage and was used to analyze the behaviour of the remaining larvae. Lastly, on days 4 and 14 after spawning, five planulae were removed from each flask (total n = 25 for each temperature regime) and photographed with a digital microscope camera to estimate their size.

Since larvae did not display clear bottom probing behaviour, the onset of competency was defined by settlement or pelagic metamorphosis. After the first larval settlement (day 14), substrate was provided to the culture flasks in order to monitor settlement behaviour. Three flasks from each temperature regime were randomly selected and three pieces (approximate diameter: 5 mm) of basalt rock attached to a plastic slide (10 mm ×80 mm) were offered as potential substrate in each flask. Basalt was selected because it is an abundant hard substrate in the deep seafloor of the Azores and where the studied species is frequently observed. The substrate was not pretreated to develop biofilm. Settled larvae were observed and photographed every 2–3 days to assess and describe settlement and metamorphosis, during a period of approximately two weeks. After metamorphosis was observed, a mixture of live microalgae (Nannochloropsis gaditana and Chaetoceros calcitrans) and rotifers was provided weekly as a potential food source.

Statistical analysis

For all the dependent variables in question, we firstly performed exploratory analysis (Zuur, Ieno & Elphick, 2010) to select the most appropriate modeling method. The effect of each independent variable was subsequently tested with linear models (LMs), by adding the independent variables progressively to the respective model and using maximum likelihood ratio (MLR) tests and the Akaike Information Criterion (AIC). Data collected from monitoring larvae stages (proportions) were modeled by means of Generalized Additive Models (GAMs) with a binomial distribution. Summarized results of the MLR test for each variable in question are provided in Table 1, while the results from each selected model are provided graphically as supplementary material (Figs. S1–S5). Statistical analysis was performed in R (R Core Team (2019)).

Spawning

Gamete release occurred for the first time on the 27th of November, one day after the new moon. Oocytes were encountered 15 min after enrichment with free live sperm, in both aquaria. Spawning was not synchronized among colonies, neither among polyps of the same colony. Despite careful observation, it was not possible to directly observe polyps releasing sperm or oocytes and determine whether one or more colonies participated in gamete release. Similarly, it was not possible to directly observe if fertilization was internal or external. All collected oocytes were fertilized, therefore fertilization was either internal, or external with very high fertilization rates. Oocytes were spherical, they had no visible germinal vesicle and were released in batches of 10-80 at a time. They were mostly negatively buoyant, however, they remained in suspension for several hours due to water movement within the aquaria. Average oocyte diameter was 365.4 ±24.2 µm. Gamete release was slow and sometimes continued for 1–3 h. It happened multiple times a day (every 2–3 h) for a week and continued with lower frequency (every 1–3 days) for approximately a month. Release occurred both during day and night hours and did not seem to follow any circadian pattern.

Embryonic and larval development

Cell division was always equal but cleavage varied highly among stages and embryos. It was not possible to determine the timing of the first division after spawning. Cytokinesis was never visible for the 2-cell stage, in which cleavage seemed to be always superficial (Fig.1B). During the following stages, cleavage varied from radial to pseudospiral and in some cases superficial, leading to embryos with substantial differences in shape. Development always led to a hollow blastula (Fig. 1G) followed by gastrulation and the formation of planula larvae without visible oral pore (Fig. 1I). Cleavage and cell division did not differ between the two rearing temperatures.

At 13 °C, all embryos reached the blastula stage within 10 h and the early gastrula stage within 48 h (Fig. 2). After 72 h all embryos reached the late gastrula stage and could perform slow, mainly rotating movements by cilia, while fully competent, swimming planulae were formed after 96 h (4 days). During their development, embryos were negatively buoyant and accumulated at the bottom of the flasks. In the first batch this resulted in the formation of embryo aggregations and abnormal embryo development. This issue was solved by adding slight aeration that ensured water and oxygen circulation within the flasks. At 15 °C, during the first 6 h cleavage seemed to be occurring at similar intervals until reaching the blastula stage (Fig. 2), however, embryos reached the late gastrula and subsequently the planula stage approximately 24 h (after 72 h) earlier than embryos reared at 13 °C (Fig. 2).

Embryos between the 2-cell and 32-cell stage obtained variable shapes (Fig. 1) and their volume was on average 0.03 ±0.0073 mm³. Subsequently, during the 64-cell stage and blastula they turned more spherical but had a similar volume range (0.03 ±0.005 mm³). After reaching the planula stage, embryos increased significantly in size (Table 1) and planulae reached 0.28 ±0.1 mm³ on day 4 and 0.67 ±0.28 mm³ on day 14, with measurements on day 14 displaying substantial variability. Mature planulae displayed the capacity to change their shape between spherical and elongated, and more elongated larvae were observed on day 14 compared to day 4 (Fig. S1). This was also confirmed from the LW ratio which presented a non-significant decrease from late gastrula embryos (1.49 ±0.17 mm³) to planulae on day 4 (1.22 ±0.29 mm³) but increased significantly (Table 1) on day 14 (2.02 ±0.45 mm³). Embryo sizes were not statistically different between the two temperatures (Table 1). Planulae on day 4 had significantly higher LW ratios at 15 °C (LW = 1.59 ±0.39; Table 1), showing a tendency to maintain a more elongated shape than at 13 °C (Fig. S1).

Embryo and larval survival

In both temperatures, survival differed substantially among batches (Fig. S2). In most batches reared at 13 °C, a sharp decline in survival rates was observed during the first 48 h, after which a more moderate mortality rate was established (Fig. 3). In the same temperature treatment, median survival time, i.e., time when mortality reached 50%, was 11 days while survival after 36 days was 16.4%. At 15 °C, the average mortality rate seemed to be more constant (Fig. 3). Median survival time was 5 days longer than at 13 °C (16 days), however, final survival after 36 days was slightly lower (12.6%). Overall, these differences were not statistically significant according to the log-rank test (p = 0.05; Fig. 3).

Swimming behaviour

Planulae remained mostly at the bottom of the culture flasks, where they displayed slight rotational and unidirectional movements. They rarely became waterborne without the aid of water movement. Once in the water column, larvae did not show a specific swimming pattern but followed random trajectories. Overall, for larvae reared under 13 °C, 51.2 ±14.2% of the recorded larval tracks were directed upwards while 50.7 ±6.33% were directed downwards. It was not clear if downward movement involved swimming or just sinking. The proportion of upward/downward swimming larvae did not change significantly with time (Table 1). Larvae displayed an average swimming speed of 0.24 ±0.16 mm s⁻¹ on day 4 and 0.36 ±0.21 mm s⁻¹ on day 15. Swimming speed did not differ significantly between upward and downward movements (Table 1) but it was significantly higher on day 15 compared to day 4 (Table 1).

Swimming velocity for larvae reared under 15 °C was similar between upward and downward swimming (Table 1) and increased slightly but significantly with time (Table 1), from 0.4 ±0.24 mm s⁻¹ on day 4 to 0.44 ±0.23 mm s⁻¹ on day 15. Overall, 52.7% of the recorded tracks were directed downwards and the proportion of upward/downward swimming tracks did not differ significantly between dates (Table 1). Larvae swimming velocity was significantly higher under 15 °C compared to 13 °C, (Fig. 4) both on day 4 and day 15 (Table 1).

Pelagic phase and settlement

The proportion of planulae decreased substantially during the course of the experiment, mainly due to high mortality (Fig. 4A). The surviving planulae followed slightly different trends between the two temperatures with planulae under 15 °C remaining in the pelagic phase for a longer period (Fig. 4B), a difference that was statistically significant (Table 1). In both temperatures, after day 36 only a minimal proportion of larvae remained (Fig. 4A) and the last free swimming planulae were observed on day 39.

Larvae started settling on day 14 under 13 °C and on day 17 under 15 °C. Under both experimental temperatures, larvae settled on the flask walls and plastic slides whereas no larvae attached to the provided basalt rock. Since the addition of substrate did not have any effect on settlement behaviour, data from all flasks, i.e., with and without provided substrate, were pooled together for further analysis. All settled larvae underwent metamorphosis. Larvae firstly obtained a pear-like shape and subsequently became rounder, gradually forming a polyp base, mouth and mesenteries (Fig. 5A). Fully developed primary polyps were formed within approximately 2–3 days, after the formation of tentacles, sclerites and tentacle pinnules (Fig. 5B). In both rearing temperatures, the number of settled larvae corresponded to a very low proportion of the initial pool of planulae, corresponding to 3.21% (11 larvae) under 13 °C and 1.46% (5 larvae) under 15 °C. Nevertheless, surviving planulae displayed slightly but significantly different trends during the course of the study (Table 1), with a larger proportion of larvae settling earlier under 13 °C than under 15 °C (Fig. 4). A high variance was observed on the estimates of the average proportion of settled larvae (Fig. 4) among batches at 13 °C, mainly due to a single batch in which very few larvae settled throughout the study period.

After day 20, an increasing proportion of the surviving larvae initiated metamorphosis without settling (Fig. 4), in both temperature regimes. This form of pelagic metamorphosis started with planula larvae obtaining a pear shape (Fig. 6A) and continued with formation of mouth, mesenteries, tentacles and finally sclerites (Figs. 6B, 6C). Metamorphosis from planula larva to primary polyp took approximately 2–3 days. None of the larvae that displayed pelagic metamorphosis settled during the course of the study. Metamorphosed larvae were still able to get transported by water movements but displayed limited swimming ability. The trend of pelagically metamorphosed larvae appeared to be significantly different between the two temperatures (Table 1), but the constructed model was heavily influenced by one batch under 15 °C in which all remaining planulae on day 31 metamorphosed pelagically and subsequently presented deformations and deceased (Fig. 4B). Overall, during the experimental period 26 larvae metamorphosed pelagically under 13 °C and 28 under 15 °C, representing only 7.5% and 8.18% of the initial planulae pool. Deformed larvae were observed in both temperatures but represented a small proportion of the initial pool (2.02% under 13 °C and 1.16% under 15 °C). Under 13 °C, they started appearing on day 24 (Fig. 4B) but remained in low numbers throughout the experimental period. Under 15 °C, they appeared 2 days later, but reached significantly higher proportions after day 35 (Table 1, Fig. 4B). Most of these late deformations under 15 °C were observed in pelagically metamorphosed larvae (Fig. 6D).